CSIR NET Life Science Unit 12

Biosensors
A biosensor is a measurement instrument that contains an immobilized
biological material (enzyme, antibody, nucleic acid, hormone, organelle,
or complete cell) that can interact with an analyte and produce physical,
chemical, or electrical signals. An analyte can be any compound like
glucose, urea, drug or pesticide whose concentration has to be measured.
Biosensors in general are analytical devices that detect changes in
biological processes and convert the data into an electrical signal. Most of
the biosensors have immobilized enzymes.

Biosensors have the following characteristics:

• Stability

• Economical

• Sensitivity

• Reproducibility

The biosensor's block diagram is divided into three sections bioreceptor,

transducer, and detector of electric signals (Figure 1).

The sensor is a sensitive biological part in the first segment, the detector part
in the second segment alters the resulting signal from the analyte contact,
and the results are shown in an accessible manner in the third segment.
The last section includes a signal conditioning circuit, an amplifier, and a
display unit, as well as the CPU. A biosensor is made up of two major
 parts: a biological component other than a cell or an enzyme, and
a physical component including an amplifier or a transducer. The
biological component communicates with the analyte to generate a signal
that can be detected by the transducer. The biological material is properly
immobilized over the transducer, and it can be utilized repeatedly for an
extended period of time.

Biosensors can be classed according to typical forms of bioreceptor

interactions including antibodies/antigens, enzymes/ligands, nucleic
acids/DNA, cellular structures/cells, or biomimetic materials, but the type
of biomolecule utilized can vary widely.

• Antigen-antibody interaction – Serological testing (covid testing)

• Artificial binding proteins - Recombinant binding fragments

(Fab, Fvor scFv) or domains (VH, VHH) of antibodies. Antigen Binding
Proteins (AgBP) are capable of selective binding to different target
proteins while keeping the parent molecule's beneficial qualities. Display
techniques such as phage display, ribosome display, yeast display, or
mRNA display are frequently used in vitro to select family members that
specifically bind to a given target antigen.

• Enzymes/ligands - Here the enzyme converts the analyte into a product

that is sensor-detectable, and enzyme inhibition or activation is detected
by the analyte or monitoring modification of enzyme properties resulting
from interaction with the analyte.

o Nucleic acids/DNA - Nucleic acid-based receptors can be either

based on complementary base pairing interactions referred to as
genosensors or specific nucleic acid-based antibody mimics
(aptamers) as aptasensors.

o Cellular structures/cells – Here for example cells of microalgae are

entrapped on a quartz microfiber and the chlorophyll fluorescence
modified by herbicides is collected at the tip of an optical fiber
bundle and transmitted to a fluorimeter.

o Microbial biosensors – These exploit the response of bacteria to a

given substance eg: arsenic detection by ars operon.

• The following characteristics are used to classify biosensors in a broad

sense:
 o Bio affinity devices - These rely on the selective binding of a
surface-restricted ligand partner (antibody, oligonucleotide, DNA,
cell) to a target analyte.

o Bio catalytic devices - Here, an immobilized enzyme is used to aid

in the identification of the target substrate. Sensor strips with
immobilized GOX, for example, have proved used for monitoring
diabetes mellitus at home.

Biosensors can also be classified based on transducer type. The most relevant
ones are noted below.

Calorimetric Biosensors

Many enzyme-catalyzed processes emit "heat," which can be detected with

biosensors (exothermic). Calorimetric biosensors assess the temperature
change in the solution containing the analyte as a result of enzyme action
and interpret the temperature change in terms of the analyte
concentration in the solution. This is the most widely used type of
biosensor, and it can detect turbid and brightly colored liquids. The most
significant disadvantage is maintaining the temperature of the sample
stream at a consistent temperature, say + 0.01°C. For most applications,
the sensitivity (10-4 M) and range (10-4 -10-2 M) of such biosensors are
insufficient. The sensitivity of the biosensor can be improved by linking
multifunctional enzymes. The use of glucose oxidase for glucose
determination is one example.

• Electrochemical transducer - Here chemical reaction between

immobilized receptors and the target analyte produces ions or electrons,
which are used to measure the electrical properties of the
solution. Amperometric and potentiometric transducers are the most
commonly used types.

1. Amperometric transducer - The current resulting from the

electrochemical oxidation or reduction of an electroactive species
is measured using amperometry. The Clark oxygen electrode, which
determines the decrease of O2 present in the sample (analyte)
solution, is used in the simplest amperometric biosensors and is
grouped under first-generation Redox reactions that can be
analyzed by amperometric biosensors. The measurement of
glucose using glucose oxidase is an example of these biosensors.
i. Enzyme-based glucose transducer - Leland C. Clark developed this
enzyme-based electrochemical biosensor. Glucose oxidase catalyzes the
oxidation of glucose to gluconolactone, which is hydrolyzed to gluconic
acid and hydrogen peroxide.

Glucose + H2O + O2 → Gluconic acid + H2O2

Glucose +Glucose oxidase (FAD) → Glucose oxidase (FADH2) + Glucanolactone

Glucose oxidase (FADH2) + O2 → Glucose oxidase (FAD) + H2O2

The enzyme glucose oxidase acts as a bioreceptor molecule and once it binds
with the glucose molecule, it oxidizes glucose to gluconic acid and
hydrogen peroxide. For the measurement of glucose in an aqueous
solution, three different transducers are used.

• An oxygen sensor for determining O2 concentration - converts O2 to

electric current.

• A pH sensor for measuring gluconic acid – converts pH change to voltage

change.

• A peroxidase sensor that measures H2O2 concentration - converts

peroxidase concentration to electric current. Biosensor is made of dialysis
membrane using oxygen electrode a thin layer of glucose oxidase
entrapped on it. In this Amperometric electrochemical glucose biosensor
the amount of glucose was calculated by reduced
dissolved O2 concentration, which is proportional to glucose
concentration. Glucose dehydrogenase instead of glucose oxidase can be
also used, but its uses are practically restricted due to the oxidation of
NAD+ to NADH, NADH increase voltage and it has to be mediated.

Product
Substrate Bioreceptor
obtained

Choline Choline oxidase H2O2

Ethanol Ethanol oxidase H2O2

 Hypoxanthine Hypoxanthine H2O2

L-glutamate Glutamate oxidase H2O2

Sucrose Invertase H2O2

Glucoamylase / glucose
Oligosaccharides H2O2
oxidase

1. Potentiometric transducer - These biosensors transform a biological

reaction into an electrical signal using ion-selective electrodes. pH meter
glass electrodes (for cations), glass pH electrodes coated with a gas
selective membrane (for CO2, NH3, or H2S), or solid-state electrodes are
the most frequent electrodes used. Many reactions generate or consume
H+, which the biosensor detects and measures; in these circumstances,
extremely weakly buffered solutions are utilized. The amount of gas
produced is detected and measured with gas sensing electrodes. Urease,
which catalyzes the processes below, is one example of such an electrode.

CO(NH2)2 + 2H2O + H+ → 2NH4+ + HCO3-

Following are examples of potentiometric biosensors.

Substrate Bioreceptor Product detected

Aspartamine L-aspartase NH3

Fats Lipase Fatty acids

Glucose Glucose oxidase Gluconic acid

Urea Urease NH4+, CO2

Nitrite Nitrite reductase NH4+

Penicillin Penicillinase H+

Sulfate Sulfate oxidase HS

Enzyme Electrodes

Enzyme electrodes are a new form of biosensor that has been devised for
amperometric and potentiometric assays of substrates such as urea,
amino acids, glucose, alcohol, and lactic acid. A given electrochemical
sensor is in close contact with a thin permeable enzyme membrane
capable of reacting with the given substrates to form the electrode. The
enzyme is implanted in the membrane and, depending on enzymatic
processes, produces O2, H+, NH4+, CO2, or other tiny molecules. The
specialized sensor picks up on this. The concentration of substrates is
determined by the magnitude of the response.

Optical Biosensors

These biosensors are capable of measuring both catalytic and affinity

processes. The products created by catalytic processes induce a change in
fluorescence or absorbance, which they monitor. Alternatively, they
assess changes in the intrinsic optical characteristics of the biosensor
surface as a result of dielectric molecules such as protein being loaded on
it (in case of affinity reactions). The firefly enzyme luciferase is an example
of this type of biosensor that uses luminescence to identify bacteria in
food or clinical samples. The bacteria are lysed to release ATP, which is
used by luciferase to create light in the presence of oxygen, which is
monitored by the biosensor.

Bio affinity Sensors

Bio affinity sensors have only lately been developed. It uses equilibrium
binding to determine the concentration of the determinants, i.e.
substrates. This demonstrates a high level of sensitivity. It uses
 radiolabelled, enzyme-labeled, or fluorescence-labeled substances
that have a wide range of properties. A receptor is radiolabeled and
permitted to bind with a determinant analog fixed on the surface of a
transducer in this biosensor. When a determinant's concentration is
raised, the tagged receptor forms an intimately bonded complex with it.

Piezoelectric biosensors

They are sometimes known as acoustic biosensors since they work on the
principle of acoustics (sound vibrations). These biosensors are made up of
piezoelectric crystals. Positive and negative-charged crystals have distinct
vibrational frequencies. The adsorption of specific molecules on the
crystal surface changes the resonance frequencies, which can be detected
using electronic instruments. These crystals can also hold enzymes with
gaseous substrates or inhibitors.

The use of acetylcholine esterase in a piezoelectric biosensor for

organophosphorus insecticides has been developed.
Formaldehyde, (biosensor) with formaldehyde dehydrogenase is another
example.

Biosensors are also classified based on various generation tools

• First-generation instruments - Here, the two components, the biocatalyst

and the transducer, can be separated and both can work without the
other.

• Second-generation instruments - Enzyme membrane electrodes that

incorporate mediators in detection are available, for example, GOX
association with peroxide detection in diluted samples.
•
Third-generation instruments - Here, the reaction creates a response
without the usage of intermediaries. The biochip can be used in such
equipment when electrochemistry happens on a semiconductor. The
progression from the use of a freely diffusing mediator (O2 or artificial) to
a system where the biocatalyst and mediator are immobilized at an
electrode surface, thereby making the recognition component an integral
part of the electrode transducer, is characterized by the third generation
sensors that do not have reagents. Redox mediator label the enzyme
followed by enzyme immobilization could be used to achieve
immobilization of enzyme and mediator.

• A successful biosensor must have some of the following features:

o High specificity/sensitivity for the analyte

o The reaction used should be independent of factors like stirring, pH,

o temperature etc.,

o The response time should be linear over a useful range of analyte

concentrations,

o Tiny and bio-compatible in case it is to be used for analyses within

the body.

o Cheap, durable, and easy to use

o Range and detection limit

Applications of Biosensor

o Uses in Medicine and Health (Glucose biosensor)

o Uses in Pollution Control (a biosensor coupled with oxygen

electrode and immobilized Trichosporon cutaneum is used for
measuring biological oxygen demand (BOD)

o Uses in Industry (spectrophotometer and auto-analyzer are used to

estimate the substrates utilized and the products formed in the
fermented broth)

o The biosensor in Military biosensor application is to provide

support to the military with such a biosensor that can detect toxic
gases including chemical warfare agents.